Storage and stability:

MyTaq™ HS Mix MyTaq HS Mix is shipped on dry/blue ice. On arrival store at -20 °C for optimum stability. Repeated
freeze/thaw cycles should be avoided.

Shipping: On Dry/Blue ice Catalog numbers: Expiry:

When stored under the recommended conditions and handled correctly, full activity of the kit is
retained until the expiry date on the outer box label.
BIO-25045: 200 x 50 L reactions 4 x 1.25 mL
Safety precautions:
Batch No.: See vial BIO-25046: 1000 x 50 L reactions 20 x 1.25 mL Please refer to the material safety data sheet for further information.
Concentration: 2x Quality control specifications:
MyTaq HS Mix and its components are extensively tested for activity, processivity, efficiency,
Store at –20 °C heat activation, sensitivity, absence of nuclease contamination and absence of nucleic acid
contamination prior to release.
Notes:
For research or further manufacturing use only.
Trademarks:
HyperLadder and MyTaq are trademarks of Bioline Reagents Ltd.

Description
MyTaq™ HS Mix is a ready-to-use 2x mix for fast, highly-specific, hot-start PCR. MyTaq HS Mix is powered by antibody mediated hot-start
and does not possess polymerase activity during the reaction set-up, thus reducing non-specific amplification. The advanced formulation of
MyTaq HS Mix allows fast cycling conditions to be used, greatly reducing the reaction time without compromising PCR specificity and
yield. Thanks to its speed and high specificity MyTaq HS Mix is also highly suitable for end point multiplex PCR. MyTaq HS Mix contains
all the reagents (including stabilizers) necessary for trouble-free PCR set up. The product is supplied conveniently all in one tube to reduce
the number of pipetting steps and to facilitate increased efficiency, throughput and reproducibility.

Components Recommended cycling conditions for colony PCR

of fragment up to 1 kb
200 Reactions 1000 Reactions
Step Temperature Time Cycles
MyTaq HS Mix, 2x 4 x 1.25 mL 20 x 1.25 mL
Initial denaturation 95 °C 1 min 1

Denaturation 95 °C 15 s
Standard MyTaq HS Mix Protocol
User
Annealing* 15 s 25-35
The following protocol is for a standard 50 L reaction and can be determined
used as a starting point for reaction optimization. Please refer to the
Important Considerations and PCR Optimization section. Extension* 72 °C 10 s

PCR set-up: * These parameters may require optimization, please refer to the Important
Considerations and PCR Optimization section if needed.

Template 200 ng
Multiplex PCR Protocol
Primers (20 M each) 1 L
MyTaq HS Mix is suitable for multiplex PCR; adjustment of the
MyTaq HS Mix, 2x 25 L cycling conditions may be required. As a starting point we
recommend using the following conditions:
Water (dH2O) up to 50 L
Recommended standard cycling conditions for multiplex PCR
* These parameters may require optimization, please refer to the Important

PCR cycling conditions: Step Temperature Time Cycles

Step Temperature Time Cycles Initial denaturation 95 °C 2 min 1

Initial denaturation 95 °C 1 min 1 Denaturation 95 °C 30 s

Denaturation 95 °C 15 s User 25*

Annealing/Extension* 4 min*
determined
User
Annealing* 15 s 25-35
determined Considerations and PCR Optimization section if needed.

Extension* 72 °C 10 s
Important Considerations and PCR Optimization
* These parameters may require optimization, please refer to the Important The optimal conditions may vary from reaction to reaction and are
Considerations and PCR Optimization section if needed. dependent on the template/primers used.

Colony PCR Protocol
MyTaq HS Mix can be used for amplification of plasmid DNA directly
from liquid cultures or from colonies on agar plates:
- From liquid culture: up to 8 μL of the overnight culture can be
directly added to the final reaction mix.
- From colonies: we recommend using a sterile tip to stab the colony
and resuspend it directly in the 50 μL reaction mix.
Primers: Forward and reverse primers are generally used at the final
concentration of 0.2-0.6 M each. As a starting point, we recommend
using a 0.4 M final concentration (i.e. 20 pmol of each primer per
50 L reaction volume). Too high a primer concentration can reduce
the specificity of priming, resulting in non-specific products.
When designing primers we recommend using primer-design
software such as Primer3 (http://frodo.wi.mit.edu/primer3) or visual
OMPTM (http://dnasoftware.com) with monovalent and divalent cation
concentrations of 10 mM and 3 mM respectively. Primers should have
a melting temperature (Tm) of approximately 60 °C.

Website: www.bioline.com/ email: info@meridianlifescience.com

PI-50152 V7
Template: The amount of template in the reaction depends mainly on
the type of DNA used. For templates with low structural complexity,
such as plasmid DNA, we recommend using 50 pg-10 ng DNA per
50 L reaction volume. For eukaryotic genomic DNA, we recommend a
starting amount of 200 ng DNA per 50 L reaction, this can be varied
between 5 ng-500 ng. It is important to avoid using template
resuspended in EDTA-containing solutions (e.g. TE buffer) since EDTA
chelates free Mg2+.
Initial denaturation: The initial denaturation step is required to activate
the enzyme and fully melt the template. We recommend 1 minute of
initial denaturation at 95 °C, however for more complex templates such
as eukaryotic genomic DNA, longer initial denaturation times of up to 3
minutes may be required.
Denaturation: Our protocol recommends a 15 s cycling denaturation
step at 95 °C, which is also suited to GC-rich templates (>55%). For low
GC content amplicons (40-45%), the denaturation step can be
decreased to 5 s.
Annealing temperature and time: The optimal annealing temperature
is dependent upon the primer sequences and is usually 2-5 °C below
the lower Tm of the pair. We recommend starting with a 55 °C annealing
temperature and, if necessary, running a temperature gradient to
determine the optimal annealing temperature. Depending on the
reaction the annealing time can also be reduced to 5 s.
Extension temperature and time: The extension step should be
performed at 72 °C. The extension time depends on the length of the
amplicon and the complexity of the template. An extension time of 10 s
is sufficient for amplicons under 1 kb or up to 5 kb for low complexity
template such as plasmid DNA. For amplification of fragments over 1kb
from high complexity template, such as eukaryotic genomic DNA, longer
extension times are recommended. In order to find the fastest optimal
condition, we suggest increasing the extension time up to 30 s/kb.
Multiplexing: When doing multiplex PCR the recommended 2-step
cycling protocol may be optimized as follows:
- Annealing/extension temperature: we highly recommend initially using
a temperature gradient to determine the optimal annealing
temperature needed for the primer set used.
- Annealing/extension time: in most cases a 4 min annealing/extension
step is largely sufficient. However in order to reduce the overall
cycling time this step can be reduced down to 1 min, especially in
the case of a lower number of multiplex amplicons.
- Cycling number: we recommend starting with 25 cycles and if
necessary, optimizing this parameter. An excess of cycles may
generate diffuse bands, too few may result in weak or no
amplification.

Troubleshooting Guide

Problem Possible Cause Recommendation

Missing component - Check reaction set-up and volumes used

- Check the aspect and the concentrations of all components as well as the storage
Defective component
conditions. If necessary test each component individually in controlled reactions
No PCR
- Decrease the annealing temperature
product - Run a temperature gradient to determine the optimal annealing temperature
Cycling conditions not optimal
- Increase the extension time, especially if amplifying a long target
- Increase the number of cycles

Difficult template - Increase the denaturation time

Excessive cycling - Decrease the number of cycles

Smearing
Extension time too long - Decrease the extension time

or Annealing temperature too low - Increase the annealing temperature

Non-Specific Primer concentration too high - Decrease primer concentration

products
- Replace each component in order to find the possible source of contamination
Contamination
- Set up the PCR and analyze the PCR product in separate areas.

Technical Support Associated Products

Product Name Pack Size Cat No
If the troubleshooting guide does not solve the difficulty you are
experiencing, please contact your local distributor or our Technical Agarose 500 g BIO-41025
Support with details of reaction set-up, cycling conditions and
Agarose tablets 300 g BIO-41027
relevant information.
HyperLadder™ 1kb 200 Lanes BIO-33025
Email: mbi.tech@meridianlifescience.com
SureClean Plus 1 x 5 mL BIO-37047

____________________________________________________________________________________________________________________________
Bioline Reagents Ltd Meridian Life Science Inc. Bioline GmbH Bioline (Aust) Pty. Ltd
UNITED KINGDOM USA GERMANY AUSTRALIA

Tel: +44 (0)20 8830 5300 Tel: +1 901 382 8716 Tel: +49 (0)3371 60222 00 Tel: +61 (0)2 9209 4180
Fax: +44 (0)20 8452 2822 Fax: +1 901 382 0027 Fax: +49(0)3371 60222 01 Fax: +61 (0)2 9209 4763

Website: www.bioline.com/ email: info@meridianlifescience.com